The activation and covalent binding of Adriamycin to DNA and microsomal proteins was studied in isolated rat liver nuclei. Evidence was gained that VP-16 is metabolized by a peroxidative mechanism involving an oxygen-centered free radical. The substrate capacity of mAMSA and mAQDI for aldehyde oxidase was determined. A method developed to monitor the activity of thymidylate synthetase in intact cells was used to study the effects of MTX and fluoropyrimidines on the flux through this enzyme. The concentrations of 5-FU and MTX required to produce cell toxicity correlated with concentrations required to inhibit thymidylate synthetase in the intact cell.